Incidence of resistance to benzimidazole fungicides in a field population of Venturia inaequalis /

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Abstract

The allele-specific polymerase chain reaction (PCR) was used to screen for
the presence of benomyl resistance, and to characterize their levels and frequencies
in field populations of Venturia inaequalis during two seasons. Three hundred
isolates of V. inaequalis were collected each season from infected leaves of MalusX
domestica. Borkh c.v. Mcintosh. The trees used were sprayed in the year prior to
collection with five applications of benomyl, its homologue Azindoyle, or water.
Monoconidial isolates of V. inaequalis were grown on 2% potato dextrose agar
(PDA) for four weeks. Each isolate was taken from a single lesion from a single
leaf. Total genomic DNA was extracted from the four week old colonies of V.
inaequalis, prepared and used as a template in PCR reactions. PCR reactions were
achieved by utilizing allele-specific primers. Each primer was designed to amplify
fragments from a specific allele. Primer Vin was specific for mutations conferring
the ben^^"^ phenotype. It was expected to amplify a 171 bp. DNA fragment from the
ben^"^ alleles only. Primers BenHR and BenMR were specific for mutations
conferring the ben"" and ben'^'' phenotypes, respectively. They were expected to
amplify 172 bp. and 165 bp. DNA fragments from the ben"" and ben"^" alleles,
respectively.
Of the 953 isolates tested, 414 (69.9%) were benomyl sensitive (ben^) and
179 (30.1%) were benomyl resistant. All the benomyl resistant alleles were ben^"",
since neither the ben"" nor the ben"" alleles were detected. Frequencies of
benomyl resistance were 23%, 24%, and 23% for the 1997 collections, and were
46%, 26% and 38% for the 1998 collections for benomyl, Azindoyle and water treatments, respectively.
Growth assay was performed to evaluate the applicability of using PCR in
monitoring benomyl resistance in fungal field populations. Tests were performed on
14 isolates representing the two phenotypes (ben^ and ben^"'' alleles) characterized
by PCR. Results of those tests were in agreement with PCR results. Enzyme
digestion was also used to evaluate the accuracy and reliability of PCR products.
The mutation associated with the ben^"'' phenotype creates a unique site for the
endonuclease enzyme Bsh^236^ allowing the use of enzyme digestion. Isolates
characterized by PCR as ben^'^'^ alleles had this restriction site for the SsA7l2361
enzyme.
The most time consuming aspect of this study was growing fungal isolates on
culture media for DNA extraction. In addition, the risk of contamination or losing the
fungus during growth processes was relatively high. A technique for extracting DNA
directly from lesions on leaves has been used (Luck and Gillings 1 995). In order to
apply this technique in experiments designed to monitor fungicide resistance, a
lesion has to be homogeneous for fungicide sensitivity. For this purpose, PCR
protocol was used to determine lesion homogeneity. One hundred monoconidial
isolates of V. inaequalis from 10 lesions (10-conidia/ lesion) were tested for their
phenotypes with respect to benomyl sensitivity. Conidia of six lesions were
homogeneous, while conidia of the remaining lesions were mixtures of ben^ and
ben^ phenotypes. Neither the ben" nor the ben' phenotype was detected.